Influenza (influenza virus) is a highly infectious acute infection of the upper and lower respiratory tracts, which is triggered by influenza viruses. Influenza occurs as a worldwide epidemic, particularly in the winter months, although individual viruses can also trigger pandemics. Current strategies to control influenza outbreaks are based on prophylactic vaccinations in combination with antiviral treatments, for example, with neuraminidase inhibitors. A majority of seasonal human influenza vaccines are inactivated trivalent split virion vaccines. These are composed of viral antigens, the envelope glycoprotein hemagglutinin (HA) and neuraminidases (NA) of three different influenza virus subtypes. The respective antigens are obtained from purified virus particles by membrane solubilization using detergents (e.g. CTAB (cetyltrimethylammonium bromide) or Tween 20 (polysorbate 20)).
In classical production processes, the influenza viruses are produced in incubated chicken eggs. In addition to the limited scalability of this process and the problems linked thereto, there are systemic difficulties in producing individual pandemic influenza viruses (e.g. H5N1) in chicken eggs to meet the growing demand of the worldwide market. Lastly, the issue of allergic reaction to chicken protein has also meant that production processes using mammalian cells in bioreactors have been established for several years. After culturing viruses in cell cultures it is necessary to separate the viruses from the contaminants (e.g. host cell proteins, DNA) so that they can be further used in their pure form. It is advantageous, furthermore, to separate infectious from non-infectious molecules or particles. In biopharmaceutical production processes, deoxyribonucleic acid (DNA), and other proteins arising from the host cell culture, occur as by-products. These generally count as contaminants and must be removed from the end product during the work-up. In the production of viruses, DNA fragments of the host cells or of the viruses themselves are also released which have to be removed.
The adsorption of viruses on solid phases by chromatographic purification has a major significance in virus purification, especially on a process scale. This is characterized in that molecules are bound to the adsorber material (ion exchanger, hydrophobic or hydrophilic adsorbents) and may then be eluted in a purified form in a subsequent step. In this case, it is important that the adsorption process proceeds reversibly so that high yields are achieved. Either a simple enrichment or a separation into two or more target substances can be carried out where, in the latter case, either the adsorption or the desorption or both can be effected selectively. The major disadvantage of the chromatographic media used for this purpose is their relatively low selectivity, and the contaminants (DNA fragments and host cell proteins) present in solution can also bind to the adsorber material such that long process development times are required in order to determine the optimal binding and elution conditions (pH, ionic strength and selection of the buffer systems).
Affinity chromatography offers enhanced selectivity. It is known from the prior art that sulfated cellulose matrices have enhanced selectivity towards influenza viruses and heparin-binding proteins.
EP 0 053 473 A1 discloses cellulose sulfate salts having heparin-like effect for use as anticoagulants. For sufficiently high anti-coagulant effect, together with desired high long-term stability under physiological conditions, the degree of sulfation (the degree of substitution of the OH groups by sulfate groups on the C2, C3 and C6 atom of the glycopyranose units) of the cellulose is between 0.8 and 2.6. The cellulose sulfate salts are obtainable by three different methods: by reaction with chlorosulfuric acid in the presence of an amine, by reaction with SO3-amide complexes in which, for example, dimethylformamide may be used as amide, or by reaction with SO3-amine complexes in which, for example, pyridine may be used as amine. In the latter variant mentioned, the cellulose is reacted with an SO3-pyridine complex at room temperature for 30 to 35 minutes and subsequently neutralized with NaOH.
EP 1 698 641 A1 discloses cellulose sulfate salts as therapeutic active ingredients for skin disorders such as atopic eczema. The cellulose sulfate salts are characterized by an inhibitory effect on hyaluronidase and have a sulfate content of 6.5 to 19.0% by weight based on the total weight of the cellulose sulfate salt. In the preparation, crystalline cellulose is firstly pre-swollen in a solvent such as pyridine, dimethyl sulfoxide or dimethylformamide. The resulting mixture is added to a sulfating reagent selected from the group of chlorosulfonic acid, piperidine-sulfuric acid complex, SO3-pyridine complex, SO3-trimethylamine complex or sulfuric acid anhydride-dimethylformamide complex, wherein the latter mentioned complex is preferred.
Both documents mentioned above, EP 0 053 473 A1 and EP 1 698 641 A1, do not disclose any chromatographic separating materials for purifying viruses. Such separating materials are commercially available as gels and are used for the purification of the molecules and viruses mentioned above. Use of these media for purifying viruses often affords low binding capacities for the viruses and low virus yields. Viruses may have sizes up to 500 nm and particulate chromatography gels with pore sizes in the range of 30 to 400 nm are therefore not very suitable. Typically, viruses can then only bind to the outer surface of the particle which explains the low binding capacities.
The sulfating of chromatographic gels composed of cellulose, dextran or agarose and the use of these sulfated gels for purifying influenza viruses are known from the prior art. By way of example, RD 298 025 A discloses affinity chromatography gels which can be prepared by sulfating “Cellulofine GH-25”, “Sepharose CL-6B” or “Sephadex G-50” with a chlorosulfonic acid-pyridine complex at 65 to 70° C., subsequent neutralization with NaOH and washing with phosphate-buffered saline solution. The gels are suitable for purifying proteins such as hepatitis B antigens, HIV-1 and HIV-3 viruses, SV40-T antigens, blood-clotting factors 7, 8, 9 and 11, nucleic acid polymerases, interferones or lysozyme.
EP 0 171 086 A2 describes a method for sulfating (partially) crystalline polysaccharide gels based on agarose, dextran or cellulose with chlorosulfonic acid or anhydrous sulfuric acid in pyridine at 65 to 70° C. The degree of sulfation of the polysaccharide gels is generally between 0.1 and 40%, whereas the degree of sulfation of the cellulose gels is specifically between 0.1 and 5%. Influenza viruses or antigens from chicken embryo cell cultures can be purified with the sulfated cellulose gels.
WO 2008/039136 A1 discloses a porous polysaccharide matrix, preferably from agarose, to which “extender” molecules of 500 kDa dextran are bound, wherein sulfate groups are in turn bound to the dextran molecules as ligands, for purifying viruses. The matrix is used for the separation of viruses, preferably influenza viruses, from DNA contaminants, in which the viruses are initially adsorbed on the matrix and are subsequently eluted with a suitable buffer. To prepare the aforementioned matrices, sulfate ligands are firstly bound to the “extender” molecules before the sulfated “extender” molecules are fixed on the polysaccharide matrix.
EP 0 171 771 A2 discloses the use of the sulfated cellulose gels known from EP 0 171 086 A2 for purifying rabies viruses from chicken embryo cell cultures.
EP 0 173 268 A2 discloses the use of the sulfated cellulose gels known from EP 0 171 086 A2 for purifying glycoproteins gA and gB as constituents of herpes simplex viruses of the HSV-1 and HSV-2 types, which are obtained from lysates of mammalian cell cultures. The glycoproteins gA and gB are purified in the presence of an anionic or non-ionic surfactant.
EP 0 171 765 A2 discloses a method for purifying Japanese encephalitis virus for vaccine production, in which the sulfated cellulose gels known from EP 0 171 086 A2 are used.
All aforementioned documents disclose chromatographic separation media consisting of particulate porous gel particles. For this purpose, polysaccharide gels having a molecular weight cut-off (MWCO) of less than 107 Da are used as base material for the sulfation reaction. Viruses, such as e.g. influenza viruses having a diameter greater than 100 nm and a molecular mass (MW) greater than 108 Da, as described by R. W. H. Ruigrok et al. in J. Gen. Virol. (1984), 65, 799-802, can only penetrate to a limited extent the pores of the porous separation materials described, in which case only a part of the binding capacity of these separation materials can be exploited.
Adsorption membranes, in contrast to particulate adsorbents, have substantially larger pores which are fully accessible to viruses. In addition, they offer the possibility to force perfusion with the medium, by applying a hydraulic pressure difference between their two main surfaces, whereby instead of a purely diffusive transport of the adsorbents in the direction of a concentration gradient into the adsorbent interior, a convective transport is achieved which can be effected much more rapidly at high flow. A further inherent disadvantage of particulate adsorbents can thereby be avoided, which is referred to as “diffusion limiting”, which consists of the fact that, with increasing particle size of the adsorbent and increasing molar mass of the adsorbent, the time required for establishing the adsorption equilibrium increases considerably, which translates into deterioration of the kinetics. For this reason, only low flow rates are typically achieved with chromatographic gels in the purification of influenza viruses for example.
Sulfated membrane adsorbers based on cellulose have already been successfully produced by sulfating a cellulose hydrate membrane, in which the sulfation is typically effected by reaction of the cellulose membrane with a Lewis base-SO3 complex in a solvent suitable for the reaction. Compared to conventional sulfated polysaccharide gels, sulfated membrane adsorbers have the following advantages:                convective flow in contrast to diffusive “flow” in particulate separating materials        higher flow rates possible        better accessibility to the ligands and higher binding capacity for viruses        simpler scalability, i.e. increasing scale from low-volume membrane adsorbers to large-volume membrane adsorbers.        
Since the capacity is a function of the ligand density and the available binding surface, this is typically increased by decreasing the membrane pore size, which can, however, have a negative effect in respect of the flow and blocking up characteristics.
U.S. Pat. No. 5,667,684 discloses microporous membranes for removing HIV viruses from blood plasma, which consist of a hydrophobic base material, e.g. polypropylene, polyethylene or polyvinylidene fluoride, onto which a chain of alkoxyalkyl acrylates, glycidyl acrylates or acrylamides have been grafted.
Sulfated cellulose is immobilized on this intermediate graft layer by covalent binding, in which, for example, free OH groups of the sulfated cellulose are reacted with functional groups, epoxide groups for example, of the repeating units of the graft chain. The HIV viruses are predominantly removed by the sulfated cellulose units.
U.S. Pat. No. 8,173,021 B2 discloses a preparation process for sulfated, non-crosslinked cellulose membranes having a degree of sulfation (sulfony group content) between 0.5 and 15%, in which cellulose membranes are reacted with a chlorosulfonic acid-pyridine complex at not more than 40° C. The chlorosulfonic acid-pyridine complex is prepared by adding chlorosulfonic acid to pyridine at a maximum of 0° C., subsequent reaction at 60° C. and cooling to a maximum of 40° C. The sulfated cellulose membranes, which can be prepared from non-crosslinked, regenerated cellulose membranes (e.g. RC-55 membranes from Whatman), are used in affinity chromatography for purifying protein-containing virus fragments or for purifying intact virus particles, which are used subsequently in influenza vaccine production.
L. Opitz et al., Biotechnology and Bioengineering, 103 (6), 2009, 1144-1154, disclose the preparation of sulfated cellulose membranes by reacting strengthened, non-crosslinked cellulose membranes with a solution of chlorosulfonic acid in pyridine at 37° C. for 12 hours. The sulfur content of the sulfated cellulose membranes, at 16 μg of sulfur/g of dried membrane, is significantly lower than in the particulate adsorbent Cellufine® Sulfate (≥700 μg of sulfur/g of dried membrane). The sulfated cellulose membranes are used for purifying three influenza strains, i.e. H1N1, H3N2 and “B/Malaysia/2506/2004” viruses, in which undesired contaminants, such as double-stranded DNA or cell constituents, are effectively removed from the cell culture solution used for the production of the viruses. With increasing NaCl concentration in the buffer solution used for the purification (150 nM versus 50 mM), the adsorption capacity of the membranes for the viruses decreases. In comparison to cation exchanger membranes, such as Sartobind® S75 or Sartobind® C75, the sulfated cellulose membranes allow an improved depletion of double-stranded DNA and permit a higher virus yield than chromatography columns based on Cellufine® Sulfate.
US 2012/0171750 A1 investigates the influence of reaction temperature on the degree of sulfation of the cellulose matrix in the preparation of sulfated cellulose matrices known from the aforementioned article by L. Opitz et al., the adsorption capacity for MVA viruses and the contamination of the purified MVA virus preparation with DNA from the cell culture solution. With increasing reaction temperature in the sulfation (35, 40 or 45° C.), the degree of sulfation of the cellulose matrix increases from 5.3 to 13% by weight, based on the weight of the cellulose polymer backbone, while the DNA fraction in the purified virus preparation increases from 7.4% to 17% and the yield of purified MVA viruses increases from 66 to 80%. The purification of the virus preparation by means of sulfated cellulose matrices may be combined with an additional step of hydrophobic interaction chromatography, in which chromatography matrices having phenyl, butyl or hexyl ligands are used.
Using the method known from U.S. Pat. No. 8,173,021 B2, a degree of sulfation greater than 15% cannot be achieved despite using relatively fine membranes (RC-55 membranes from Whatman with a pore size of 0.45 μm). The selected pore size is also unfavourable for binding viruses having a diameter of more than 100 nm, since loss of flow and blocking up of the membrane during the adsorption of the viruses can occur.